This invention relates generally to novel immunosuppressive agents that inhibit calcineurin function, methods for identifying immunosuppressive agents, and treatments for immune system related diseases and conditions.
While immune reactions are required to protect animals from deleterious foreign antigens, certain immune reactions can result in pathological conditions, e.g., autoimmune disease, allergies, transplant graft rejection and graft-versus-host disease.
Calcineurin plays a pivotal role in the T cell receptor (TCR)-mediated signal transduction pathway, leading to the transcriptional activation of certain cytokines, e.g., IL-2, which are required for an immune response. Calcineurin is a calcium and calmodulin-dependent protein serine/threonine phosphatase. Calcineurin has been shown to modulate the activity of several transcription factors that bind to the IL-2 promoter, including NF-AT, NF-xcexaB and AP-1. It has been reported that calcineurin dephosphorylates the cytoplasmic subunit of NF-AT, allowing it to translocate into the nucleus to activate transcription.
The immunosuppressive drugs cyclosporin A and FK506 target calcineurin, inhibiting its protein phosphatase activity. These drugs, however, can exhibit nephrotoxic and neurotoxic effects after long term usage. There is a need for additional immunosuppressive agents which can inhibit calcineurin function.
It is an object of the invention to provide a novel immunosuppressive agent.
It is another object of the invention to provide a novel immunosuppressive agent that binds to calcineurin.
It is yet another object of the invention to provide a novel immunosuppressive agent that inhibits interaction between calcineurin and NF-AT.
It is yet another object of the invention to provide a method for inhibiting an immune reaction using a novel immunosuppressive agent that inhibits calcineurin function.
According to the invention, an isolated polypeptide, called Cabin 1, comprising the amino acid sequence as set forth in SEQ ID NO:1, or biologically active analogs thereof, is provided. In certain embodiments, the Cabin 1 polypeptide, or biologically active analogs thereof, is capable of binding to calcineurin and/or inhibiting calcineurin function.
Another aspect of the invention is an isolated polypeptide fragment of Cabin 1 polypeptide which is capable of binding to calcineurin and/or inhibiting calcineurin function, or biologically active analogs thereof. In preferred embodiments, the polypeptide fragment of Cabin 1 is a C-terminal fragment of the Cabin 1 polypeptide. For example, the invention includes fragments comprising the amino acid sequence as set forth in SEQ ID NO:2 (Cabin 1-54), SEQ ID NO:3 (Cabin 1-6), SEQ ID NO: 4 (Cabin 1-97) and SEQ ID NO:5 (Cabin 1-14). Other preferred fragments include SEQ ID NO:6 (Cabin 1-CNBD23) and SEQ ID NO:7 (Cabin1-CNBD 13).
Another aspect of the invention is an isolated polynucleotide comprising a polynucleotide encoding the polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, or biologically active analogs thereof. In certain embodiments, the polynucleotide comprises the nucleotide sequence as set forth in SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14 or SEQ ID NO: 15.
Another aspect of the invention is a gene therapy vector comprising a nucleotide sequence encoding Cabin 1 polypeptide or a fragment thereof capable of binding to calcineurin or inhibiting calcineurin function, or a biologically active analog thereof. In certain preferred embodiments, the fragment is a C-terminal fragment of Cabin 1.
Another aspect of the invention is a cell having a gene therapy vector described herein.
Another aspect of the invention is a method for producing a polypeptide capable of binding to calcineurin and/or inhibiting calcineurin function, comprising culturing a cell having a gene therapy vector described herein under conditions that permit expression of the polypeptide.
Another aspect of the invention is a method for treating an immune-related disease or condition in an animal. A gene therapy vector described herein is administered to the animal.
Another aspect of the invention is a method for inhibiting an immune response in an animal. An animal in need of inhibition of an immune response is provided. A therapeutically effective amount of a Cabin 1 polypeptide or a fragment thereof capable of binding to calcineurin or inhibiting calcineurin function, or a biologically active analog thereof, is provided. In certain preferred embodiments, the fragment is a C-terminal fragment of Cabin 1. The polypeptide or fragment, or biologically active analog thereof, is administered to the animal so as to inhibit the immune response in the animal. In certain embodiments, the polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or biologically active analogs thereof.
Another aspect of the invention is a composition for treating an immune-related disease or condition in an animal comprising a therapeutically effective amount of Cabin 1 polypeptide or a fragment thereof capable of binding to calcineurin or inhibiting calcineurin function, or a biologically active analog thereof, and a pharmaceutically acceptable carrier.
Another aspect of the invention is a composition for treating an immune-related disease or condition in an animal, comprising a therapeutically effective amount of a recombinant nucleic acid having a nucleotide sequence encoding Cabin 1 polypeptide or a fragment thereof capable of binding to calcineurin or inhibiting calcineurin function, or a biologically active analog thereof, and a pharmaceutically acceptable carrier.
Another aspect of the invention is a method of inhibiting protein phosphatase activity by calcineurin. Calcineurin and a substrate, e.g., NF-AT, for calcineurin phosphatase activity are provided. Cabin 1 polypeptide or a fragment thereof capable of inhibiting protein phosphatase activity by calcineurin, or a biologically active analog thereof, is provided. The calcineurin and polypeptide or fragment, or biologically active analog thereof, are contacted such that the protein phosphatase activity by the calcineurin on the substrate is inhibited. In certain embodiments, the inhibiting is in vitro. In certain embodiments, the inhibiting is in vivo
Another aspect of the invention is a method for identifying an agent that is immunosuppressive. A cell is provided. An agent, e.g., a fragment of Cabin 1 polypeptide, a biologically active analog thereof, or a mimetic thereof, is provided. The agent is contacted with the cell. The effect of the agent on an aspect of calcineurin function in the cell is evaluated. A change in the aspect of calcineurin function is indicative of the agent being immunosuppresive. In certain preferred embodiments, the aspect of calcineurin function is protein phosphatase activity, e.g., dephosphorylation of NF-AT. In certain preferred embodiments, the aspect of calcineurin function is binding of calcineurin to Cabin 1 polypeptide or a fragment thereof.
Another aspect of the invention is a method for identifying an agent that is immunosuppresive. Calcineurin and NF-AT are provided. An agent, e.g., a fragment of Cabin 1 polypeptide, a biologically active analog thereof, or a mimetic thereof, is provided. The agent is contacted with the calcineurin. The effect of the agent on dephosphorylation of the NF-AT by the calcineurin is evaluated. A change in the dephosphorylation is indicative of the agent being immunosuppressive.
Another aspect of the invention is a method for evaluating an agent for use in treating a disease involving an immune reaction resulting in pathology. A cell, cell-free system or animal is provided. An agent is provided. The agent is administrated to the cell, cell-free system or animal in a therapeutically effective amount. The effect of the agent on an aspect of Cabin 1 metabolism is evaluated. A change in the aspect of Cabin 1 metabolism is indicative of the usefulness of the agent in treating a disease involving an immune reaction resulting in pathology.
Another aspect of the invention is an isolated antibody, e.g., monoclonal or polyclonal, which selectively binds to an antigenic determinant of a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
The above and other features, objects and advantages of the present invention will be better understood by a reading of the following specification in conjunction with the drawings.